Current Organic Chemistry - Volume 8, Issue 5, 2004

The present issue of Current Organic Chemistry, “ Complex Carbohydrates” consists of six reviews on complex carbohydrate structures that modify proteins and lipids. The authors of these articles are leading researchers in the areas of glycan structure / function relationships and have performed pioneering studies on glycoproteins, proteoglycans and glycosphingolipids. The first review by Professor G.W Hart's group is entitled “Nucleocytoplasmic Glycosylation, O-linked β-NAcetylglucosamine” and describes the nature, localization, structure and possible roles for O-GlcNAc modifications in specific cellular pathways including signal transduction, nuclear transport, transcription, protein metabolism, apoptosis and functions performed by cytoskeletal proteins. This review further describes the interplay of the O-GlcNAc modification with phosphorylation. The authors also deal with the implications of the O-GlcNAc modification in dysregulated cellular processes leading to hyperglycemia, obesity, type II-diabetes, cancer and neurodegeneration. The second review by Professor M.C. Glick's group is concerned with “The Glycobiology of Cystic Fibrosis”. The review focuses on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in pulmonary epithelial cells. The CFTR, a glycoprotein acting as a Cl- channel is extensively modified by repeating units of N-acetyllactosamine. The glycosylated CF airway epithelial cell exhibits a glycophenotype characterized by increased expression of fucosyl residues and / or decreased expression of sialic acid. This review further elaborates on the possibility of compartmental differences in the Golgi between CF and non-CF glycosyltransferases for the generation of CF glycophenotypes. The third article, entitled “Relationships of Connective Tissue Glucosaminoglycans and Proteoglycans” is by Professor J.E. Silbert, who describes the nature and structure of connective tissue proteoglycans containing the linear polysaccharide (glucosaminoglycan) hyaluronan, glucosaminoglycan chondroitin / dermatan sulfate and keratan sulfate. Matrix proteoglycans are classified into two groups, namely the “hyalectins” and the SLRP (small leucine-rich proteins). Furthermore, the review provides information about the structure, biosynthesis, degradation and turnover, classification, distribution and function of different glycans in connective tissue glucosaminoglycans and proteoglycans. In addition to their glycosylation, the phosphorylation and sulfation of such macromolecules is also considered within the context their biosynthesis. The fourth review by Professors Ph. Roussel and P. Delmotte deals with “ The Diversity of Epithelial Secreted Mucins ” in human, amphibian and fish epithelia. These mucins are heavily glycosylated, wherein the O-linked glycans make up to 80% of the mucin molecular mass. Furthermore, multimerization of mucins via disulfide bridges occurs during biosynthesis and generates biomolecules of highest complexity. The review also covers the general properties, diversity in types and post-translational modifications of secreted mucins in different vertebrate subgroups. The secand last article by Professor D.C. Hoessli and co-workers is entitled “Glycosphingolipid Clusters as Organizers of Plasma Membrane Rafts and Caveolae”. This review described the contribution of glycosphingolipids in the organization of plasma membrane rafts and caveolae, thus building up a new vision of membrane organization and function. For instance, the role of rafts and caveolae in signal transduction, adhesion, endocytosis and entry of microbial pathogens is discussed. Lastly, the use of rafts and caveolae as therapeutic targets in the treatment of metabolic diseases and cancer is discussed. The last article by Professor Nasir-ud-Din deals with “Combinatorial Metabolism in Plasmodium falciparum-infected Erythrocytes and Interplay and Glycosylation and Phosphorylation”. The review proposes the concept of combinatorial metabolism in the P. falciparun-infected erythrocyte and develops this concept regarding the glycosylation and phosphorylation of MSP-1 and other parasitic proteins, a process in which catalysis of such modifications is carried out by host-cell enzymes acting upon parasitic proteins. Furthermore, the functional consequences of one amino acid modification by a glycan or a phosphate are discussed for specific parasitic proteins.

O-linked β-N-acetylglucosmaine (O-GlcNAc) is an essential, ubiquitous, dynamic modification of metazoan nucleocytoplasmic proteins. Unlike prototypical glycosylation, O-GlcNAc is not elongated into more complex structures and it is localized almost exclusively to nuclear and cytoplasmic proteins. O-GlcNAc modifies Ser / Thr residues in peptide motifs either identical or similar to those used by kinases. In some instances, O-GlcNAc and phosphorylation occur at the same site, suggesting that a complex interplay exists between these post-translational modifications. Deletion of the gene that adds O-GlcNAc to the protein backbone, the UDP-GlcNAc: polypeptide O-β-Nacetylglucosaminyltransferase, is lethal at the single cell level underlying the importance of O-GlcNAc. O-GlcNAc is rapidly emerging as a key nutrient sensor regulating signaling, transcription and cellular responses to stress.

Cystic fibrosis (CF), the most common lethal genetic disease of Caucasians, is characterized by pathology to the exocrine organs, especially the lungs which are the site of most of the morbidity and mortality of the disease. The gene causing CF was defined more than a decade ago and a deletion of F508 has been shown to be the most common mutation. The gene product the cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel on the apical surface of epithelial cells and has been extensively studied. It is a large glycoprotein but the glycosylation has not yet been defined. On the other hand, examination of glycosylation on the surface of CF airway epithelial cells has shown a glycophenotype, which is usually expressed as increased fucosyl residues and / or decreased sialic acid. Studies have shown a direct relationship of CFTR to the CF glycophenotype in airway epithelial cells. The terminal glycosyltransferases which are responsible for the CF glycophenotype have been studied. A difference in these enzymes and the mRNA expression was not sufficient to account for the altered surface glycoproteins of CF and non CF cells. It may be possible that a compartmental difference in the Golgi between CF and non CF glycosyltransferases may be responsible for the CF glycophenotype.

Connective tissue matrix contains the linear polysaccharide (glycosaminoglycan) hyaluronan, and the glycosaminoglycans chondroitin / dermatan sulfate, and keratan sulfate covalently linked to protein (proteoglycans). Hyaluronan is a linear polymer consisting of alternating N-acetylglucosamine and glucuronic acid residues; chondroitin / dermatan has alternating N-acetylgalactosamine and glucuronic or iduronic acid residues; keratan has galactose and N-acetylglucosamine residues. There are two classes of matrix proteoglycans named “hyalectin” and “SLRP (small leucine-rich protein).” Hyalectins have similar large core proteins and large numbers of glycosaminoglycan chains, while SLRPs have similar small core proteins with few glycosaminoglycan chains. Synthesis proceeds by modification of glucose to form specific sugar nucleotides that act as the direct precusors for forming the glycosaminoglycans and linkage oligosaccharides on specific amino acids of specific proteins. The chondroitin / dermatan and keratan are modified by variable sulfation and in the case of dermatan, by epimerization of some of the glucuronic to iduronic. These modifications provide for multiple structural variations that in turn provide a high degree of specificity in function. In general, the functions of matrix hyaluronan and the matrix proteoglycans relate to interactions with other matrix substances such as collagen to provide scaffolding and shape. Their variable structures provide specificity of function for particular tissues, and specific aspects of interaction with other matrix molecules.

Secreted mucins are a family of polydisperse, high molecular mass and highly glycosylated glycoproteins synthesized by mucosae, or skin of amphibians and fish. Most secreted mucins are synthesized in goblet cells of epithelial surfaces or mucous cells of exocrine glands. They are probably the most complicated biological molecules ever described. Except for a low molecular mass species, the peptide part of mucin, or apomucin, is very long and can contain from # 5,000 to more than 13,000 amino acids. In man, the apomucins synthesized in goblet or mucous cells are encoded by four mucin genes (MUC), organized in a cluster. Apomucins are covered by hundreds of carbohydrate chains. Their biosynthesis implicates different glycosylation processes, mostly O-glycosylation, which are responsible for about 80 % of the mucin molecular mass. During their biosynthesis, secreted mucins may dimerize and multimerize via disulfide bridges. The carbohydrate chains resulting from the O-glycosylation process are extremely diverse and contribute to the possible formation of different mucin glycoforms from a single apomucin peptide. In man, the glycosylation differs from one individual to another according to histo-blood bood groups (ABO, secretor, Lewis...). Moreover, there are large differences in the O-glycosylation processes between different animal species. Preliminary data suggest that different factors endogeneous (cytokines and / or hormones), and exogeneous (microbial), are involved in the regulation of mucin biosynthesis. Owing to their physical properties, secreted mucins have an essential role in the protection of underlying epithelia. Mice genetically deficient in one of the mucin genes (MUC2) frequently develop tumors. Finally, the wide diversity of carbohydrate chains, specific for a given species, is very important in specific recognition phenomena (interactions with microbes, fertilization...).

Glycosphingolipids aggregate with sphingomyelin and cholesterol in the outer leaflet of the plasma membranes to become included in transmembrane domains comprising saturated phospholipids in the inner leaflet, select transmembrane proteins and acylated proteins anchored to the cytofacial aspect. Such domains constitute liquid-ordered, planar and short-lived rafts, floating in the liquid-disordered glycerophospholipid medium of the membrane outer leaflet. When scaffolded by the protein caveolin, such rafts assume the shape of microscopically detectable small invaginations or caveolae. Rafts and caveolae are plasma membrane microdomains involved in membrane trafficking, endocytosis, transcytosis, signal transduction and adhesion, and may be opportunistically utilized by various microbial pathogens to enter many different types of host cells. By virtue of the greater cohesion of their sphingolipid and cholesterol components, rafts and caveolae constitute stable membrane platforms that associate receptors, kinases, phosphatases and adaptor proteins, and promote their interactions in a membrane environment conducive to optimal signaling. Such membrane platforms are involved in a variety of cellular interactions with extracellular matrices, cells, microbes and soluble ligands. The capacity of protein receptors to associate with or dissociate from rafts is in part due to the affinity of their “lipid shells” for the sphingolipid aggregates, as well as to their extramembranous properties and characteristics. In particular, carbohydrate side-chains of N-glycosylated receptors may interact with gangliosides in rafts and then change the conformation, affinity and signaling properties of the glycoprotein receptors. Rafts and caveolae are targeted in several therapeutic approaches aiming at modifying their contents and function. Such approaches have been applied successfully to the treatment of cancer cells and of cells unresponsive to physiological insulin triggering.

The nature of protein modifications, particularly surface glycosylation, in Plasmodium falciparum has long been observed, but in recent times controversial reports appeared. It is considered that the surface modifications, O- and N-glycosylation, have been of particular importance to the malarial parasite itself as well as to the erythrocyte. The modifications in the erythrocytic membranes and changes in the parasitic structure have been the subject of intense investigations in recent years. Comprehensive elaborations resulted in the development of means to describe the extensively modified structural and antigenic properties of the host cell and parasitic molecules that are of vital significance in the understanding of the functions of biologically important molecules such as the merozoite surface protein-1 (MSP-1). A combinatorial behavior of the parasitic and host cell molecules becomes the basis for molecular modifications of the parasite proteins and the events leading to invasion and survival of the parasite. The MSP-1 protein is modified on the surface by a sugar moiety O-glycosidically linked in β-configuration. This β-O-GlcNAc modication of MSP-1 has been predicted by computer assistance and experimentally verified. The Plasmodium falciparum proteins are known to be phosphorylated, and phosphorylation of MSP-1 has been predicted; moreover, there is sufficient experimental evidence to suggest phosphorylation. Computer assisted studies suggest a strong possibility of interplay between glycosylation and phosphorylation in proteins with β-O-GlcNAc modification.